Oncogenic STAT3 is an attractive therapeutic target given its role in cancer development and metastatic progression (1). Although mutations in STAT3 are rarely observed (2), aberrantly activated STAT3 has been implicated in the pathogenesis of most cancers including leukemia (2), head and neck squamous cell carcinoma (HNSCC; ref. 3), colon cancer (4), melanoma (5), breast, and others (6). These effects are principally mediated through the transcriptional regulation of genes involved in proliferation, apoptosis, and invasion of tumor cells as well as modulation of the tumor microenvironment. The mechanisms of persistently activated STAT3 include abnormal cytokine or growth factor signaling through the Janus kinases, receptor tyrosine kinases [RTK; e.g., EGF receptor (EGFR)], and non-RTKs (e.g., Src and BCR-ABL; ref. 7). Given that many tumor types seem to be “addicted,” in a sense, to STAT3 hyperphosphorylation for their survival and proliferation and that STAT3 loss in normal adult tissue is dispensable, the demonstration of an efficacious STAT3 inhibitor would be of great therapeutic value for many malignancies.

Despite a plethora of data implicating STAT3 as the leading culprit in the pathogenesis of several tumor types, transcription factors such as STAT3 have traditionally been deemed “undruggable.” Tyrosine kinase inhibitors that target upstream receptor/non-receptor kinases including EGFR (8), JAK (9), or Src (10) inhibitors lead to inhibition of STAT3 phosphorylation and consequently, modulation of signaling downstream of STAT3, and therefore have served as surrogate STAT3 inhibitors. Although STAT3 knockdown using RNA interference approaches have served to show a requirement for STAT3 in tumor growth both in vivo and in vitro, such approaches are at best, preclinical and would be difficult to translate into effective therapeutic tools (11).

Now, a new study by Sen and colleagues in this issue of Cancer Discovery (12) discusses the development and efficacy of a novel type of STAT3 inhibitor, an oligodeoxynucleotide decoy with high specificity toward phosphorylated STAT3. Transcription factors generally bind to specific consensus motifs in promoter regions of target genes and thus initiate a cascade of events downstream that result in proliferation, survival, or differentiation. An oligodeoxynucleotide decoy works by binding with higher affinity to these target regions and subsequently renders the protein incapable of binding to their actual targets. Figure 1 shows the mechanism of binding of STAT3 oligodeoxynucleotide decoy to STAT3 target sequences. The E2F family of transcription factors was the first to be targeted for therapeutic purposes (treatment of coronary bypass grafts prevented restenosis) using such an approach (13). Following the success of this approach, scientists have devised oligodeoxynucleotide decoys for a variety of other transcription factors including NF-κB, AP-1, and CREB (14). However, a significant limitation to their effectiveness is their short half-life and relative instability.

Mechanism of action of a transcription factor decoy to STAT3. A, activated or tyrosine phosphorylated STAT3 dimerizes and translocates to the nucleus where it binds to specific motifs on target sequences. B, when an oligonucleotide decoy to STAT3 is injected, it is incorporated via endocytosis and sequesters activated dimeric STAT3, thus competing with sequences in the DNA, blocking transcriptional output.

The STAT3 decoy was developed as a double stranded 15-mer oligonucleotide whose sequence was derived from the STAT3 response element in the c-fos promoter and binds competitively for STAT3. This binding blocks the expression of various STAT3 target genes including Bcl-xL and cyclin D1. The STAT3 decoy was tested in several preclinical models showing efficacy and lack of toxicity in vivo, thus laying the groundwork for a phase 0 clinical trial (15).

There are several points of interest in this study. This study is the first phase 0 clinical trial in HNSCCs that included a vehicle treatment arm. The addition of the vehicle-treated arm was crucial in determining the effectiveness and specificity of the decoy. Furthermore, the accessibility of these head and neck tumors makes them a very attractive model system to evaluate the efficacy of the decoy at the site of injection and in adjacent tissue including normal cells. The authors first determined the kinetics of downregulation of STAT3 target genes in xenograft and non-human primate models (15). On the basis of these results, they injected the STAT3 decoy directly into the patients' tumor immediately before surgery and approximately 4 hours later assessed their molecular effects following resection of the tumor. Direct intratumoral injections of the decoy caused robust modulation of target gene expression in the patient tumors that received the decoy versus those who received the vehicle treatment. Interestingly, the authors failed to detect any association between baseline levels of either phosphorylated or total STAT3 with degree of target gene downmodulation, suggesting that even elevated levels of phosphorylated or total STAT3 in tumor tissues can be targeted with a single administration of the decoy. These results warrant further studies in HNSCCs and other STAT3-dependent tumors.

It is tempting to speculate whether an autocrine/feed forward loop or a “field effect” was occurring in the tumors of those patients that responded to the decoy. For example, reducing the function of STAT3 in a few cells could lead to both apoptosis and reductions in growth factor production resulting in a “ripple-effect” promoting cell death in neighboring cells. Gene expression analyses of tumor and surrounding “normal” tissue or laser microdissection would shed further light on the interplay between tumor and stromal interactions in the setting of STAT3 decoy administration. The authors do not mention any data about the uptake efficiency of the decoy. However, modifications to the decoy, so that it can now be “tracked” in vivo, would be of significant interest and importance allowing for a determination of organ tropism, intracellular penetration, optimal dosing, and regimen, and provide correlative information about response to therapy.

Given the encouraging results from the phase 0 trial but cognizant of the limitations of this labile decoy, the authors then developed more stable versions of the decoy that could potentially be administered systemically. Indeed, intravenous injections of the unmodified oligonucleotide failed to elicit any antitumor responses or effects on STAT3 target gene expression. Systematic chemical modifications to the decoy through its cyclization using hexaethylene glycol linkers on both 5′ and 3′ ends significantly increased the half-life of the decoy in animal studies compared with the parental unmodified version. Sen and colleagues (12) then used these modified oligonucleotides in their established model systems to compare the efficacy of the unmodified oligonucleotide to the modified versions. The authors found that the modified decoys had a much longer half-life in serum than in the unmodified oligonucleotide. Furthermore, the modified STAT3 decoy could bind very efficiently to phosphorylated STAT3 and at similar nanomolar affinities as the unmodified decoy. Next, the authors showed that the modified decoys could inhibit proliferation of HNSCC cell lines while interfering with expression of STAT3 target genes. Finally, the authors evaluated the efficacy of the modified decoys in an in vivo xenograft system. Treatment with the decoy by intravenous administration for 19 consecutive days reduced expression levels of cyclin D1 and Bcl-xL in the xenograft tissue, without altering levels of total or phosphorylated STAT3. The authors did not report any toxicity related with the cyclic decoy administration. Intriguingly, they found that 20% of the xenograft tumors showed complete tumor regression, suggesting that the immune system/non–cell-autonomous factors may be involved in tumor regression.

This report warrants the development of clinical-grade cyclic STAT3 decoy for an expanded phase I trial in patients with head and neck cancer who have failed standard therapy. The ability to analyze tumor tissue pre- and postinjection will lead to the establishment of molecular and cellular correlates predictive of response to therapy. Importantly, targeting STAT3 using this novel therapeutic agent is applicable to a broad range of other malignancies that are “addicted” to or dependent upon activated STAT3.